DESCRIPTION: (Applicant's Abstract) Central and peripheral tolerance mechanisms help to establish and maintain donor-host tolerance after allogeneic bone marrow transplantation (BMT). While mechanisms in central tolerance have been extensively studied, the mechanisms that mediate peripheral tolerance after allogeneic BMT are not as well defined. We have identified in murine allogeneic BM chimeras a population of thymus-derived Thy1+ abTCR+CD4+ cells of donor origin that suppress alloreactivity following infusion of donor T cells (DLI therapy) post-BMT. These "regulatory" T cells are derived from donor marrow and mature in the host thymus. We hypothesize that they are analogous to the regulatory CD4+ T cells that mediate dominant peripheral tolerance against self-antigens in intact mice. Just as others have shown that the loss of CD4+ regulatory cells may lead to autoimmune disease, we have shown that depletion of thymus-derived CD4 regulatory T cells from allogeneic BM chimeras prior to DLI therapy results in exacerbated graft-vs-host disease (GVHD). We propose to use the models of DLI therapy that we developed to further characterize the Thyl +abTCR+CD4+ regulatory cells, to investigate the afferent and efferent mechanisms by which they mediate suppression, and to evaluate the ability of ex vivo expanded regulatory T cells to suppress alloreactivity in vivo. To accomplish these goals, four Specific Aims are proposed: In Specific Aim 1, we will test the hypothesis that the CD4+ regulatory T cells from allo-BMT chimeras (like those in autoimmune models) co-express CD25 (IL2Ra) through adoptive transfer experiments with purified CD25 and CD25 cells and by using CD25 knockout mice (deficient in regulatory T cells) as BM donors. In Specific Aim 2 we will investigate afferent and efferent mechanisms of suppression. The proposed experiments build on substantial preliminary data that suggest a role for FasL on the regulatory cells (but not Fas on the DLI cells) and implicate IL1O (but not IL4) and diminished IL2 secretion as mechanisms. We will determine the cellular requirements for induction of regulatory activity, including the role of antigen-presenting cells (APCs) and costimulation. In Specific Aim 3, we will address the antigen-specificity of the regulatory T cells. We will test the hypothesis that the regulatory cells require the ongoing presence of host APCs by "parking" the cells in immunodeficient mice of host and donor origin. We will activate and expand regulatory T cells ex vivo in Specific Aim 4 and determine whether they suppress acute and/or chronic GVHD in vivo after adoptive transfer. We speculate that these adoptively transferred cells may prevent the autoimmune-like manifestations of chronic GVHD by controlling improperly selected, autoreactive donor T cells generated de novo after MHC-mismatched BMT. The proposed studies take advantage of the unique murine BMT/DLI models that we have developed and apply new insights into regulatory T cell biology gained from studies in autoimmune diseases. The information obtained will contribute significantly to our understanding of the role of regulatory T cells in mediating dominant peripheral tolerance after allo-BMT and could lead to translational applications in clinical BMT.